Human Trophoblast Progenitor Cells Express and Release Angiogenic Factors

Trophoblast stem cells develop from polar trophectoderm and give rise to the cells that generate the placenta. Trophoblast cells are responsible for the uterine invasion and vascular remodeling during the implantation of the embryo. However this knowledge is not yet to be confirmed for trophoblast progenitor cells (TPCs). In this study, we aimed to demonstrate that human TPCs (hTPCs) express and release angiogenic factors for the first time. TPCs were isolated from the term placenta. Then immunophenotyping was performed by FACS method by analyzing caudal type homeobox 2 (CDX2) and eomesodermin (EOMES). Immunofluorescence staining of CDX2 and EOMES was performed on these cells. Lastly, angiogenesis-related proteins were detected by western blot and ELISA assays. The isolated cells were positive for trophoblast stem cell markers CDX2 and EOMES in 92.5% and 92.7% of cells, respectively showing the characteristics of TPCs. The investigation of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2) at protein and mRNA level in comparison with human umbilical vein endothelial cells (HUVECs), revealed that human TPCs (hTPCs) have higher levels of VEGF and VEGFR1 transcripts. Additionally soluble forms of VEGF and VEGFR1 were detected in supernatants of hTPCs. With this information, TPCs seem to be promising for regenerative cell therapies by increasing angiogenesis.

Due to being discarded immediately after birth, the placenta is an easily accessible organ, and a good candidate as a source for regenerative medicine because of a high level of stem cells content. Importantly, all of these specialized trophoblast cells can be derived from self-renewing, multipotent cells referred to as trophoblast stem cells. Trophoblast stem cells first isolated from mouse blastocytes (2), and from human placenta  (4,5). Today, CDX2 and eomes transcription factors are mainly accepted as the trophoblast stem cell markers (6,7). In addition to these factors, fibroblast growth factor receptor 2 (FGFR2) and bone morphogenetic protein 4 (BMP4) are other known trophoblast stem cell markers (4). In 2016, Genbacev introduced pieces of evidence that integrin alpha 4 (ITGA4) was the highest level expressed factor in trophoblast stem/progenitor cells isolated from term placenta (8). Therefore, a high level of ITGA4 expression on the surface of the cells, can be used to identify TPCs. Vascular endothelial growth factor (VEGF) is one of the most important and effective angiogenesis-promoting molecules. It is an endothelial cell-specific mitogen, and initiates signal transduction through two high affinity receptor tyrosine kinases, VEGFR-1 (or FLT-1) and VEGFR-2 (or KDR/FLK-1). Additionally, these two receptors exist also as soluble forms. Soluble form of VEGFR 1 (sVGFR1) is an antagonist of VEGF action, and decreases the level of free VEGF through strong binding to it (9). In contrast to the action of sVEGFR1, sVEGFR2 does not efficiently antagonize the binding of VEGF and does not inhibit vascular endothelial growth factor A (VEGFA)-induced mitogenesis. Instead, sVEGFR2 contains the VEGF-C binding site and traps VEGF-C, and disables its binding and activation of VEGFR3, thus inhibiting lymphangiogenesis (10).
Although little is known about trophoblast stem and progenitor cells, mesenchymal stem cells (MSCs) are considerably effective for therapeutic angiogenesis in ischemia animal models as well as clinical vascular diseases (11). Previously, many studies revealed that both adult bone marrow MSCs (BMSCs) and adipose tissue-derived MSCs (AMSCs) could induce therapeutic angiogenesis (12). In addition to this wide variety of cell source,

Immunophenotyping of cells
The marker phenotype of these hTPCs was   After stopping the reaction, the plate was read at 450 nm using a spectrophotometer.

Statistical analyzes
Data were expressed as mean ±SEM. One way ANOVA test was used to compare the means of multiple groups. One Way Anova was performed using the SigmaStat 3.5 software. A value of p ≤0.05 was considered as statistically significant.

Western blot band densities were measured with the
Alpha DigiDoc 1000 gel documentation unit.
Densitometric data obtained from western blot and 2 -ΔΔCt values obtained from Q-PCR analysis were subjected to statistical analysis using the SigmaStat 3.5 software. One way Anova was performed and p≤0.05 was considered as statistically significant.
For ELISA analysis, protein concentrations were evaluated and p≤0.05 was considered as statistically significant.

Isolated cells bear the characteristics of hTPCs
hTPCs showed fibroblast-like phenotype at passage 3, and were characterized with cell surface markers by flow cytometry analysis of CDX2 and EOMES. The percentages of cell surface markers were 92.5% for CDX2 (Fig.1a, b) and 92.7% for EOMES (Fig.1c, d). Morphologically, hTPCs showed a spindle or triangular phenotype, and adhered to plastic in the second passage ( Fig.1e and f). Immunofluorescence staining of the cells provided further evaluation. It has been shown that isolated cells expressed trophoblast cell markers CDX2 (Fig.1g, h and i) and EOMES (Fig.1j, k and l).

hTPCs express and release angiogenic factors
Immunoblotting results prove that hTPCs express VEGF, VEGFR1, and VEGFR2. In comparison to positive control HUVEC, hTPCs have lower VEGF and VEGFR1 protein level and elevated VEGFR2 protein level (Fig. 2).

Fig. 4. Soluble forms of VEGF and VEGFR1 proteins in supernatants of hTPCs.
Soluble forms of VEGF and VEGFR1 were present in supernatants of hTPCs (Fig. 4). ELISA study demonstrated that the high level of sVEGFR1 was released from hTPCs to media. The soluble form of VEGFR2 in hTPC supernatants could not be detected.  In the present study, we displayed that TPCs may induce angiogenesis in transplantation era which enhances the recovery process. Therefore, they seem to be strong candidates for recruitment therapy. Many question marks still hang on TPCs topic so this topic, needs further researches.